Many genes in volved in the antio idant response, including Nrf2, were found within the group of genes show ing most deregulated e pression when MSC0 was com pared with tMSC. Since Nrf2 binds ARE containing sequences we used a previously generated list of genes known to contain selleck chemicals llc ARE in their promoters and performed GSEA with different pairs of MSC lines. This analysis showed an enrichment of ARE containing genes in those cell lines e pressing fewer number of oncogenes, e cept for the comparison between MSC4 and tMSC that showed no enrichment. We focused on the last steps during MSC transfor mation where significant changes in intracellular ROS levels were found. qRT PCR e periments confirmed down regulation of Nrf2 and selected antio idants and ARE containing genes when tMSC were compared with MSC3 and MSC4.

One of the most powerful antio idants and a major redo buffering mechanism in the cell is the glutathione system. E pression of genes involved in glutathione biosynthesis such as glutamate cysteine ligase catalytic and modifier subunits, and glutathi one synthetase fluctuated during the process of MSC transformation. We also found dimin ished e pression of glutathione reductase in tMSC, suggesting that ineffi cient conversion of o idized glutathione to its re duced form occurs in tumor cells. Concurring with these results, tMSC showed the lowest levels of the active form of glutathione, the form of glutathione able to provide antio idant power. Overall, these data indicate that transformation of MSC leads to a global transcriptional down regulation of the cellular antio idant program.

Nrf2 is repressed during cellular transformation via activation of RAS RAF ERK pathway Western blot e periments confirmed suppression GSK-3 of Nrf2 e pression and its downstream target NQO1 that correlated with ST and H RasV12 induced activation of ERK and AKT pathways. To investigate the mechanism of Nrf2 repression during transformation, we focused in the last transformation step where the more pronounced down regulation of Nrf2 and ARE containing genes occurred. We studied the roles of RAS and some RAS downstream effectors by e pressing con stitutive active mutants of H RAS, RAF 1, and AKT in immortal MSC4. We found that activation of RAS and RAF, but not AKT, led to decreased e pression of Nrf2 and NQO1. Recent reports showed that Nrf2 e pression was de creased in certain human breast cancer cells and breast tumors when compared with normal mammary epithe lial cells or normal breast tissue.